Abstracts

Rationale: Channelopathies underlie several neurological disorders, particularly those associated with excessive neural excitability such as epilepsy. Recent work has demonstrated that mutations in the gene encoding the chloride channel subtype CLCN2 are associated with certain human generalized epilepsies, including absence epilepsy (1). While it remains unknown how chloride channel dysfunction leads to such epilepsies, altered neuronal excitability within substructures of the thalamus, particularly the reticular thalamic nucleus (RT), can promote neural activity patterns characteristic of absence epilepsy. As previous studies have shown that CLCN2 expression in RT is high (2), we tested the hypothesis that CLCN2 dysfunction in RT causes hyperexcitability and promotes epileptiform activity. Methods: We examined chloride channel function using both genetic and pharmacological manipulations. In a first series of experiments, we compared the activity of RT neurons recorded in wild type mice to that of mice deficient in CLCN2 (ie CLCN2 knock mice, generous gift of James Melvin, U. Rochester). Individual RT neurons in horizontal brain slices of mice (postnatal day 11-14) were recorded using whole-cell patch-clamp electrodes. Spontaneous excitatory (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) were isolated and recorded. The instantaneous frequency and amplitude of individual currents were measured. In a second set of experiments, chloride channel regulation of RT neuron excitability was evaluated by blocking CLCN2 channels in wild type brain slices. We compared sEPSCs and sIPSCs generated in control conditions (ACSF only) to that observed during bath application of the CLCN2 antagonist NPPB (100uM). Results: Preliminary EEG studies indicate that CLCN2 knockout mice express the electrophysiological signature (ie spike and wave activity) of absence seizures. Our slice experiments indicated RT neuron hyperexcitability is a likely candidate mechanism for the generation of these seizures. Specifically, spontaneous synaptic activity of RT neurons was altered by inactivation of CLCN2. The frequency but not amplitude of sEPSCs recorded in CLCN2 knockout mice was higher than in wild type mice. In contrast, sIPSCs were indistinguishable in recordings of RT neurons from wild type versus knockout mice. Pharmacological blockade of CLCN2 yielded results consistent with the knockout mouse data. Specifically, the frequency (but not amplitude) of sEPSCs was increased during NPPB application. Also, sIPSCs were unaffected by NPPB. Conclusions: Our results reveal a potential link between chloride channel dysfunction and generalized seizure activity. Specifically, these data show that CLCN2 deficiency increases synaptic excitation in RT, and suggest that altered excitability of RT may play a mechanistic role in generation of the absence seizures. 1. Haug,K. et al. (2003). Nat Genet 33, 527-532. 2. Smith,R.L. et al. (1995). J Neurosci 15, 4057-4067.